*Target (protein/gene name): NADH-dependent fumarate reductase *NCBI Gene # or RefSeq#: *Protein ID (NP or XP #) or Wolbachia#: 11602
*Organism (including strain):Trypanosoma brucei
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 – Parasitic Agenthttp://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Essentiality of this protein:**http://jb.asm.org/content/190/21/7170.long** The protein NADH-dependent fumarate reductase is essential to the functioning of the cellular redox balance. Without this protein the cell wouldn’t be able to reduce its target compound possibly compromising the integrity of the tricarboxylic acid cycle in respiration.
Complex of proteins?: The protein fumarate reductase works with other proteins in a complex to accomplish the tasks of the tricarboxylic acid cycle. After forming the succinate in the reaction another enzyme then comes in and uses the product formed in the next step of the process.
--Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Information regarding the spectrophotometric boundaries, and running the assay are in the following two links.
-- List cost and quantity of substrate reagents and supplier Catalog #: 47910-5G, Price: $33.60, Supplier: Sigma-Aldrich
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB Query 394 LSAAIEAAGCGAQVVLMEKEAKLGGNSAKATSGINGWGTRAQAKASIVDGGKYFERDTYK 453 SAAI A GA+V+L+EKE +GGN+ A G+N T Q I D + DT KSbjct 139 FSAAISATDSGAKVILIEKEPVIGGNAKLAAGGMNAAWTDQQKAKKITDSPELMFEDTMK 198 Query 454 SGIGGNTDPALVKTLSMKSADAIGWLTSLGVPLTVLSQLGGHSRKRTHRAPDKKDGTPLP 513 G N DPALVK LS S D++ W+T++G LT + +GG S R HR P G Sbjct 199 GGQNIN-DPALVKVLSSHSKDSVDWMTAMGADLTDVGMMGGASVNRAHR-PTGGAG---- 252 Query 514 IGFTIMKTLEDH-VRGNLSGRITIMENCSVTSLLSETKERPDGTKQIRVTGVEFTQAGSG 572 +G +++ L D+ V+ N+ R+ T + K+ K I V G+ GSbjct 253 VGAHVVQVLYDNAVKRNIDLRMN-------TRGIEVLKDDKGTVKGILVKGMY-----KG 300 Query 573 KTTILADAVILATGGFSNDKTADSLLREHAPHLVNFPTTNGPWATGDGVKLAQRLGAQLV 632 + ADAVILATGGF+ + + L P L F +TN P A GDG+ +A+ G L Sbjct 301 YYWVKADAVILATGGFAKNNERVAKLD---PSLKGFISTNQPGAVGDGLDVAENAGGALK 357 Query 633 DMDKVQLHPTGLINPKDPANPTKFLGPEALRGSGGVLLNKQGKRFVNELDLRSVVSKAIM 692 DM +Q HPT + + EA+RG+G +L+N++GKRFVNE+ + S AI+Sbjct 358 DMQYIQAHPTLSVK-------GGVMVTEAVRGNGAILVNREGKRFVNEITTKDKASAAIL 410 Query 693 EQGAEYPGSGGSMFAYCVLNAAAQKLFGVSSHEFYWKKMGLFVKADTMRDLAALIGCPVE 752 Q +G S AY + + + +K S + +G+ AD++ L + G +Sbjct 411 AQ------TGKS--AYLIFDDSVRKSL---SKIDKYIGLGVAPTADSLVKLGKMEGIDGK 459 Query 753 SVQQTLEEYERLSTSQRSCPITRKSVYPCVLGTKGPYYVAFVTPSIHYTMGGCLI 807 ++ +T+ Y L +S + R ++ P L +G YY VTP +H+TMGG +ISbjct 460 ALTETVARYNSLVSSGKDTDFERPNL-PRAL-NEGNYYAIEVTPGVHHTMGGVMI 512 ---- Query Coverage: 37%
---- Max % Identities:55%
---- % Positives: 50%
---- Chain used for homology: 1JRY
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): N/A
Purification Method: Partial release from subcellular compartment by treatment with digitonin.
Image of protein (PyMol with features delineated and shown separately): (This is an image of the protein that is being compared, NOT the original protein as it could not be found on the PDB website.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): My protein sequence:
MVDGRSSASIVAVDPERAARERDAAARALLQDSPLHTTMQYATSGLELTVPYALKVVASA
KI Homology model protein sequence:
ADNLAEFHVQNQECDSCHTPDGELSNDSLTYENTQCVSCHGTLAEVAETTKHEHYNAHASHFPGEVACTS
CHSAHEKSMVYCDSCHSFDFNMPYAKKWLRDEPTIAELAKDKSERQAALASAPHDTVDVVVVGSGGAGFS
AAISATDSGAKVILIEKEPVIGGNAKLAAGGMNAAWTDQQKAKKITDSPELMFEDTMKGGQNINDPALVK
VLSSHSKDSVDWMTAMGADLTDVGMMGGASVNRAHRPTGGAGVGAHVVQVLYDNAVKRNIDLRMNTRGIE
VLKDDKGTVKGILVKGMYKGYYWVKADAVILATGGFAKNNERVAKLDPSLKGFISTNQPGAVGDGLDVAE
NAGGALKDMQYIQAHPTLSVKGGVMVTEAVRGNGAILVNREGKRFVNEITTKDKASAAILAQTGKSAYLI
FDDSVRKSLSKIDKYIGLGVAPTADSLVKLGKMEGIDGKALTETVARYNSLVSSGKDTDFERPNLPRALN
EGNYYAIEVTPGVHHTMGGVMI
*length of your protein in Amino Acids: 1142aa
Molecular Weight of your protein in kiloDaltons using the**Expasy ProtParam** website: 123689.8 kiloDaltons
Molar Extinction coefficient of your protein at 280 nm wavelength: 117450
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) Websites used: (others are listed under specific sections)
*NCBI Gene # or RefSeq#:
*Protein ID (NP or XP #) or Wolbachia#: 11602
*Organism (including strain):Trypanosoma brucei
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 – Parasitic Agenthttp://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Essentiality of this protein: **http://jb.asm.org/content/190/21/7170.long**
The protein NADH-dependent fumarate reductase is essential to the functioning of the cellular redox balance. Without this protein the cell wouldn’t be able to reduce its target compound possibly compromising the integrity of the tricarboxylic acid cycle in respiration.
Complex of proteins?: The protein fumarate reductase works with other proteins in a complex to accomplish the tasks of the tricarboxylic acid cycle. After forming the succinate in the reaction another enzyme then comes in and uses the product formed in the next step of the process.
Druggable Target: fumarate + NADH + H+
http://www.brenda-enzymes.org/literature/lit.php4?e=1.3.1.6&r=390844
*EC#: 1.3.1.6
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.3.1.6
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Information regarding the spectrophotometric boundaries, and running the assay are in the following two links.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/fluka/47910?lang=en®ion=US
-- link (or citation) to paper that contains assay information
http://www.brenda-enzymes.info/literature/lit.php4?e=1.3.1.6&r=656280
-- List cost and quantity of substrate reagents and supplier
Catalog #: 47910-5G, Price: $33.60, Supplier: Sigma-Aldrich
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Query 394 LSAAIEAAGCGAQVVLMEKEAKLGGNSAKATSGINGWGTRAQAKASIVDGGKYFERDTYK 453 SAAI A GA+V+L+EKE +GGN+ A G+N T Q I D + DT KSbjct 139 FSAAISATDSGAKVILIEKEPVIGGNAKLAAGGMNAAWTDQQKAKKITDSPELMFEDTMK 198 Query 454 SGIGGNTDPALVKTLSMKSADAIGWLTSLGVPLTVLSQLGGHSRKRTHRAPDKKDGTPLP 513 G N DPALVK LS S D++ W+T++G LT + +GG S R HR P G Sbjct 199 GGQNIN-DPALVKVLSSHSKDSVDWMTAMGADLTDVGMMGGASVNRAHR-PTGGAG---- 252 Query 514 IGFTIMKTLEDH-VRGNLSGRITIMENCSVTSLLSETKERPDGTKQIRVTGVEFTQAGSG 572 +G +++ L D+ V+ N+ R+ T + K+ K I V G+ GSbjct 253 VGAHVVQVLYDNAVKRNIDLRMN-------TRGIEVLKDDKGTVKGILVKGMY-----KG 300 Query 573 KTTILADAVILATGGFSNDKTADSLLREHAPHLVNFPTTNGPWATGDGVKLAQRLGAQLV 632 + ADAVILATGGF+ + + L P L F +TN P A GDG+ +A+ G L Sbjct 301 YYWVKADAVILATGGFAKNNERVAKLD---PSLKGFISTNQPGAVGDGLDVAENAGGALK 357 Query 633 DMDKVQLHPTGLINPKDPANPTKFLGPEALRGSGGVLLNKQGKRFVNELDLRSVVSKAIM 692 DM +Q HPT + + EA+RG+G +L+N++GKRFVNE+ + S AI+Sbjct 358 DMQYIQAHPTLSVK-------GGVMVTEAVRGNGAILVNREGKRFVNEITTKDKASAAIL 410 Query 693 EQGAEYPGSGGSMFAYCVLNAAAQKLFGVSSHEFYWKKMGLFVKADTMRDLAALIGCPVE 752 Q +G S AY + + + +K S + +G+ AD++ L + G +Sbjct 411 AQ------TGKS--AYLIFDDSVRKSL---SKIDKYIGLGVAPTADSLVKLGKMEGIDGK 459 Query 753 SVQQTLEEYERLSTSQRSCPITRKSVYPCVLGTKGPYYVAFVTPSIHYTMGGCLI 807 ++ +T+ Y L +S + R ++ P L +G YY VTP +H+TMGG +ISbjct 460 ALTETVARYNSLVSSGKDTDFERPNL-PRAL-NEGNYYAIEVTPGVHHTMGGVMI 512
---- Query Coverage: 37%
---- Max % Identities:55%
---- % Positives: 50%
---- Chain used for homology: 1JRY
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): N/A
Purification Method: Partial release from subcellular compartment by treatment with digitonin.
Image of protein (PyMol with features delineated and shown separately): (This is an image of the protein that is being compared, NOT the original protein as it could not be found on the PDB website.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
My protein sequence:
MVDGRSSASIVAVDPERAARERDAAARALLQDSPLHTTMQYATSGLELTVPYALKVVASA
DTFDRAKEVADEVLRCAWQLADTVLNSFNPNSEVSLVGRLPVGQKHQMSAPLKRVMACCQ
RVYNSSAGCFDPSTAPVAKALREIALGKERNNACLEALTQACTLPNSFVIDFEAGTISRK
HEHASLDLGGVSKGYIVDYVIDNINAAGFQNVFFDWGGDCRASGMNARNTPWVVGITRPP
SLDMLRNPPKEASYISVISLDNEALATSGDYENLIYTADDKPLTCTYDWKAKELMKPSQS
NIAQVSVKCYSAMYADALATACFIKRDPAKVRQLLDGWRYVRDTVRDYRVYVRENERVAK
MFEIATEDAEMRKRRISNTLPARVIVVGGGLAGLSAAIEAAGCGAQVVLMEKEAKLGGNS
AKATSGINGWGTRAQAKASIVDGGKYFERDTYKSGIGGNTDPALVKTLSMKSADAIGWLT
SLGVPLTVLSQLGGHSRKRTHRAPDKKDGTPLPIGFTIMKTLEDHVRGNLSGRITIMENC
SVTSLLSETKERPDGTKQIRVTGVEFTQAGSGKTTILADAVILATGGFSNDKTADSLLRE
HAPHLVNFPTTNGPWATGDGVKLAQRLGAQLVDMDKVQLHPTGLINPKDPANPTKFLGPE
ALRGSGGVLLNKQGKRFVNELDLRSVVSKAIMEQGAEYPGSGGSMFAYCVLNAAAQKLFG
VSSHEFYWKKMGLFVKADTMRDLAALIGCPVESVQQTLEEYERLSTSQRSCPITRKSVYP
CVLGTKGPYYVAFVTPSIHYTMGGCLISPSAEIQMKNTSSRAPLSHSNPILGLFGAGEVT
GGVHGGNRLGGNSLLECVVFGRIAGDRASTILQRKSSALSFKVWTTVVLREVREGGVYGA
GSRVLRFNLPGALQRSGLSLGQFIAIRGDWDGQQLIGYYSPITLPDDLGMIDILARSDKG
TLREWISALEPGDAVEMKACGGLVIERRLSDKHFVFMGHIINKLCLIAGGTGVAPMLQII
KAAFMKPFIDTLESVHLIYAAEDVTELTYREVLEERRRESRGKFKKTFVLNRPPPLWTDG
VGFIDRGILTNHVQPPSDNLLVAICGPPVMQRIVKATLKTLGYNMNLVRTVDETEPSGSS
KI
Homology model protein sequence:
ADNLAEFHVQNQECDSCHTPDGELSNDSLTYENTQCVSCHGTLAEVAETTKHEHYNAHASHFPGEVACTS
CHSAHEKSMVYCDSCHSFDFNMPYAKKWLRDEPTIAELAKDKSERQAALASAPHDTVDVVVVGSGGAGFS
AAISATDSGAKVILIEKEPVIGGNAKLAAGGMNAAWTDQQKAKKITDSPELMFEDTMKGGQNINDPALVK
VLSSHSKDSVDWMTAMGADLTDVGMMGGASVNRAHRPTGGAGVGAHVVQVLYDNAVKRNIDLRMNTRGIE
VLKDDKGTVKGILVKGMYKGYYWVKADAVILATGGFAKNNERVAKLDPSLKGFISTNQPGAVGDGLDVAE
NAGGALKDMQYIQAHPTLSVKGGVMVTEAVRGNGAILVNREGKRFVNEITTKDKASAAILAQTGKSAYLI
FDDSVRKSLSKIDKYIGLGVAPTADSLVKLGKMEGIDGKALTETVARYNSLVSSGKDTDFERPNLPRALN
EGNYYAIEVTPGVHHTMGGVMI
*length of your protein in Amino Acids: 1142aa
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 123689.8 kiloDaltons
Molar Extinction coefficient of your protein at 280 nm wavelength: 117450
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGGTAGACGGGCGATCTTCTGCATCAATTGTTGCCGTTGATCCCGAAAGGGCTGCGCGT
GAGCGCGACGCAGCAGCGCGTGCCCTTCTTCAAGACAGTCCGCTACACACGACCATGCAA
TATGCAACGTCTGGTCTTGAGCTTACCGTTCCCTACGCACTTAAGGTGGTTGCCAGTGCT
GACACCTTCGATCGCGCTAAGGAGGTTGCCGATGAGGTGCTACGCTGCGCATGGCAACTC
GCCGACACCGTGTTGAACAGTTTCAACCCGAACAGTGAGGTTTCACTCGTGGGTCGCCTG
CCTGTGGGGCAGAAGCACCAAATGTCTGCTCCACTCAAGCGTGTGATGGCATGCTGCCAG
CGTGTGTATAACTCATCGGCTGGATGTTTTGATCCCTCCACAGCACCCGTCGCAAAGGCG
CTGCGTGAGATTGCACTGGGGAAGGAGCGGAACAATGCTTGTCTGGAGGCACTTACTCAA
GCGTGTACGCTTCCCAACAGTTTTGTGATCGATTTCGAAGCTGGAACTATCAGCCGTAAG
CACGAGCATGCGTCTCTGGACCTAGGTGGGGTTAGCAAAGGTTATATCGTTGATTATGTC
ATTGATAATATCAATGCTGCTGGATTTCAAAACGTTTTTTTTGACTGGGGTGGAGACTGC
CGTGCGAGTGGTATGAACGCGCGCAATACCCCGTGGGTTGTTGGTATAACTCGCCCTCCG
TCCCTTGATATGCTCCGCAACCCGCCAAAGGAAGCGTCGTATATCAGCGTTATCTCTCTC
GACAACGAGGCCCTTGCCACGAGTGGCGATTATGAAAACTTAATATACACCGCTGATGAT
AAACCCCTTACCTGCACTTATGACTGGAAGGCGAAGGAACTGATGAAACCTTCTCAGTCC
AATATCGCGCAGGTATCGGTTAAATGTTATAGCGCCATGTACGCTGACGCGCTTGCGACT
GCGTGTTTCATAAAGCGGGATCCCGCGAAGGTTCGACAGCTGCTGGACGGTTGGCGTTAC
GTGCGTGATACAGTGAGAGATTACAGGGTCTACGTTCGTGAAAATGAGCGAGTAGCGAAG
ATGTTTGAAATCGCCACAGAGGATGCGGAAATGAGGAAGAGGCGGATCAGCAACACACTT
CCCGCTCGTGTCATTGTGGTGGGCGGTGGTCTCGCGGGTTTGTCCGCGGCCATCGAAGCT
GCAGGATGCGGTGCTCAGGTTGTGCTTATGGAGAAGGAGGCGAAGCTCGGAGGCAACAGC
GCCAAGGCGACATCTGGTATCAACGGATGGGGCACACGTGCTCAGGCGAAGGCAAGCATT
GTGGATGGTGGGAAATACTTCGAGCGTGACACATACAAGTCTGGTATCGGGGGTAACACC
GATCCTGCCCTTGTGAAGACACTTTCTATGAAAAGTGCTGACGCTATTGGGTGGCTGACC
TCGTTGGGTGTACCGCTGACGGTATTGTCACAGCTTGGGGGTCACAGCCGCAAGCGCACA
CATCGGGCACCGGATAAGAAAGATGGTACACCTCTACCTATCGGATTTACAATCATGAAA
ACCCTCGAGGATCACGTGCGTGGTAACCTTTCTGGCCGCATCACCATAATGGAAAACTGC
AGTGTAACGTCGTTGCTCAGTGAGACGAAGGAACGGCCAGATGGCACTAAACAGATACGA
GTTACTGGTGTGGAGTTCACGCAGGCTGGCAGTGGGAAGACGACCATACTTGCAGATGCT
GTCATCCTTGCCACTGGTGGATTTTCTAACGACAAAACTGCAGACTCCCTGCTTCGTGAG
CACGCCCCGCACTTGGTCAACTTCCCTACGACGAATGGCCCGTGGGCGACAGGTGATGGC
GTGAAACTTGCACAGCGACTTGGCGCTCAACTGGTGGATATGGACAAGGTCCAGTTGCAT
CCGACAGGCCTCATCAACCCGAAGGATCCAGCGAACCCTACAAAGTTCCTTGGACCTGAG
GCGCTACGTGGATCCGGTGGCGTTTTGTTAAACAAGCAAGGCAAGCGCTTCGTTAATGAA
CTTGACCTCCGTTCTGTGGTATCGAAAGCCATCATGGAACAGGGTGCGGAATATCCTGGA
TCGGGTGGTAGCATGTTCGCCTACTGTGTGTTGAATGCTGCGGCGCAGAAGCTCTTTGGT
GTCAGCTCACACGAGTTCTACTGGAAGAAGATGGGTCTCTTCGTGAAGGCTGACACCATG
AGGGACCTCGCTGCACTCATTGGGTGCCCAGTGGAATCTGTGCAGCAGACGCTGGAGGAG
TACGAGCGGCTCTCCACATCACAGCGTTCCTGCCCCATCACGCGCAAGAGCGTCTATCCG
TGCGTGCTCGGCACTAAGGGCCCCTACTACGTCGCCTTCGTGACACCTTCGATTCACTAC
ACAATGGGTGGATGTCTCATCTCGCCTTCTGCTGAAATACAAATGAAGAACACATCATCA
CGCGCTCCACTGAGTCACAGCAACCCAATCCTCGGGTTATTTGGTGCCGGTGAGGTAACG
GGTGGTGTGCACGGTGGGAACCGGTTGGGCGGCAATTCGCTGCTTGAGTGCGTCGTGTTT
GGGAGAATTGCGGGTGATCGGGCCTCGACCATCCTTCAGAGGAAGTCCTCAGCACTTTCC
TTCAAGGTGTGGACGACCGTGGTGCTGCGTGAAGTACGCGAAGGTGGTGTGTACGGTGCT
GGGTCCCGCGTGCTTCGCTTTAATTTACCCGGGGCGCTGCAACGGTCTGGTCTGAGCCTC
GGCCAATTTATCGCAATTCGTGGTGATTGGGACGGTCAGCAGTTGATCGGTTATTACAGT
CCCATCACGCTGCCAGATGATCTTGGCATGATCGATATACTCGCCCGCAGTGATAAGGGG
ACGCTGAGGGAGTGGATTTCCGCTCTGGAGCCGGGTGACGCTGTGGAGATGAAGGCATGC
GGTGGTCTGGTGATTGAGCGCCGCTTAAGCGATAAGCACTTTGTGTTCATGGGACACATT
ATCAACAAGCTTTGTCTAATTGCTGGTGGAACGGGTGTGGCACCGATGCTGCAAATAATC
AAAGCAGCCTTTATGAAACCCTTCATTGACACATTGGAGAGCGTTCATCTCATCTATGCC
GCGGAGGACGTAACGGAGTTGACGTATCGCGAGGTGCTGGAGGAGCGCCGTCGTGAGTCA
CGTGGAAAGTTCAAGAAAACGTTTGTCCTCAACCGGCCCCCGCCCCTATGGACTGATGGT
GTTGGCTTCATCGACCGGGGCATCCTCACAAATCATGTGCAGCCGCCATCTGACAACCTG
CTGGTGGCCATATGCGGACCACCGGTAATGCAGCGCATTGTAAAGGCGACCCTGAAGACT
TTGGGCTACAACATGAACCTTGTGAGGACTGTGGATGAAACGGAGCCGAGCGGCTCATCC
AAAATTTGA
Sequence Length: 3429 bp
*GC% Content for gene: 54%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Websites used: (others are listed under specific sections)
RESOURCE PAGES:
http://www.tdrtargets.org/targets/view?gene_id=11602
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://tdrtargets.org/
http://www.ncbi.nlm.nih.gov/gene
http://blast.ncbi.nlm.nih.gov/